Methods of analyzing fluorescent-labelled biomolecules after separating based on size- or charge are central to molecular biology. Examples of methods utilizing fluorescent-labelled nucleic acids include automated DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the like. In the case of multi-color automated DNA sequencing, labelled nucleic acid fragments of varying size are separated by electrophoresis, typically in a single electrophoresis lane, channel, or capillary. Employing these methods, automated four-color Sanger-type DNA sequencing has enabled entire genome characterization at the molecular level.
Stereochemical purity is of importance in the field of pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit chirality (U.S. Pat. No. 6,075,024). A case in point is provided by the L-form of the beta-adrenergic blocking agent, R(−) albuterol, which is known to be 100 times more potent than the D-enantiomer (U.S. Pat. No. 5,760,090). Furthermore, optical purity is important since certain isomers may actually be deleterious rather than simply inert.
Atropisomers are stereoisomeric conformations of a molecule whose interconversion is slow enough to allow separation and isolation under predetermined conditions (McGraw-Hill Dictionary of Chemical Terms, (1984), S. Parker, Ed., p. 36). The energy barrier to thermal racemization may be determined by the steric hindrance to free rotation of one or more bonds forming a chiral axis. Certain biaryl compounds exhibit atropisomerism where rotation around an intraannular bond lacking C2 symmetry is restricted. The free energy barrier for enantiomerization is a measure of the stability of the intraannular bond with respect to rotation. Optical and thermal excitation can promote racemization, dependent on electronic and steric factors (Tetreau (1982) Nouv. Jour. de Chimie, 6:461–65).
Ortho-substituted biphenyl compounds may exhibit this type of conformational, rotational isomerism known as atropisomerism (Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., pp. 1142–55). Such biphenyls are enantiomeric, chiral atropisomers where the sp2-sp2 carbon-carbon, intraannular bond between the phenyl rings has a sufficiently high energy barrier to free rotation, and where substituents X≠Y and U≠V render the molecule asymmetric. The steric interaction of X—U, X—V, and/or Y—V, Y—U is large enough to make the planar conformation an energy maximum. Two nonplanar, axially chiral enantiomers, shown below, then exist as atropisomers when their interconversion is slow enough such that they can be isolated free of each other. By one definition, atropisomerism is defined to exist where the isomers have a half-life t½ of at least 1000 seconds, which is a free energy barrier of 22.3 kcal mol−1 (93.3 kJ mol−1) at 300K (Oki, M. (1983) “Recent Advances in Atropisomerism,” Topics in Stereochemistry, 14:1). Bold lines and dashed lines in the figures shown below indicate those moieties, or portions of the molecule, which are sterically restricted due to a rotational energy barrier. Bolded moieties exist orthoganally above the plane and dashed moieties exist orthogonally below the plane of the rest of the molecule.

Xanthene dyes have important applications as detectable fluorescent labels of nucleic acids (U.S. Pat. Nos. 5,188,934; 5,654,442; 5,885,778; 6,096,723; 6,020,481; 5,863,727; 5,800,996; 5,945,526; 5,847,162; 6,025,505; 6,008,379; 5,936,087; 6,015,719). Xanthene compounds containing an asymmetric biannular bond can exist in stable atropisomeric forms. Conjugates of atropisomeric xanthene compounds and chiral substrates, such as nucleotides, polynucleotides, polypeptides, and carbohydrates, form diastereomers. These diastereomeric conjugates can separate under certain conditions, such as electrophoresis, chromatography, and other methods. Separation of diastereomers can hinder detection by display of double peaks or bands, i.e. “peak doubling”. Thus, atropisomerically enriched or purified forms of xanthene dyes are important as labels for methods based on separation and detection of analytes.